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M Bendayan

Publications and source records attributed to M Bendayan.

At least 199 records · Page 11Linked to original sources
PubMed1981

Application of the protein A-gold technique for electron microscopic demonstration of polypeptide hormones.

The protein A-gold (pAg) technique enables the electron microscopic detection of antigenic material on thin sections of aldehyde-fixed and Epon-embedded tissue. Application of this technique to the endocrine pancreas and gastrointestinal mucosa using antisera against insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin, and neurotensin allowed these polypeptides to be localized in the secretory granules of the corresponding specific cell types. The small size of marker gold particles permitting the precise identification of the labeled organelles coupled to the high specificity of the binding represent further evidence that the protein A-gold technique can be used as a general cytochemical reagent for the visualization of antigen-bound antibodies on thin sections.

Animals↗
PubMed1981

Hyperglycemia and microangiopathy in the eel.

When female eels, fasting and sexually mature, were progressively adapted to cold water (2-4 degrees C), their blood sugar concentration rose to values averaging 600 mg/dl. Control eels, kept in warm water (18-20 degrees C), had a mean blood sugar concentration of 100 mg/dl. After a period of 5-6 mo, the blood capillaries of the rete mirabile in the swimbladder were examined in both control, low blood sugar eels, and in cold-adapted, high blood sugar eels. In the latter, the basal laminae of the capillaries were thickened; their amino acids composition was altered and the in vitro glucose carbon incorporation into basal laminae glycoproteins was increased over a wide range of medium glucose concentrations. Furthermore, the diffusion capacity of the rete, as measured with tracer molecules during steady-state conditions in a countercurrent perfusion system, was increased in the hyperglycemic eel. It is concluded that chronic hyperglycemia in the cold-adapted eel is associated with a microangiopathy characterized by morphologic, biochemical, and functional alterations.

Adaptation, Physiological↗
PubMed1980

Use of the protein A-gold technique for the morphological study of vascular permeability.

The protein A-gold immunocytochemical technique has been applied for the ultrastructural study of vascular permeability. A capillary preparation was perfused for 30 min with a buffer solution containing albumin. After fixation and embedding, the albumin molecules were revealed on the tissue sections using anti-albumin antiserum and the protein A-gold complex. A specific labeling was obtained over the capillary lumen, the vesicular structures of the endothelial cells, and the intercapillary space. These results allowed us to conclude that the protein A-gold technique is suitable and of general applicability for the morphological study of vascular permeability.

Air Sacs↗
PubMed1980

Quantitative immunocytochemical localization of pancreatic secretory proteins in subcellular compartments of the rat acinar cell.

The recently developed protein A-gold technique for the detection of intracellular antigenic sites on thin sections was utilized to localize nine different secretory proteins in the rat exocrine pancreas. Amylase, chymotrypsinogen, trypsinogen, lipase, elastase, carboxypeptidases A and B, RNase and DNase, were detected at the level of the rough endoplasmic reticulum, the Golgi area, and the zymogen granules of the acinar cells, as well as in the acinar lumen. A quantitative evaluation of the labeling showed that its intensity was not identical for all enzymes studied nor in all cellular compartments analyzed. An increasing gradient of the labeling from the rough endoplasmic reticulum to the Golgi and to the zymogen granules was found for amylase, carboxypeptidases A and B, chymotrypsinogen, trypsinogen, and RNase, while a comparable low degree of labeling in the Golgi apparatus and in the zymogen granules was observed for DNase, lipase, and elastase. These results suggest that the nine enzymes are processed through the same intracellular compartments, but that they may be concentrated to different degrees in the zymogen granules before being released in the acinar lumen.

Animals↗
PubMed1980

Protein A-gold complex for postembedding staining of intracellular antigens.

The use of protein A-gold complex for electron microscopic demonstration of intracellular antigenic sites in thin sections of glutaraldehyde and Epon-embedded tissue is reported. The method is carried out in two steps: thin sections are incubated with specific antisera and the antigen-antibody complexes are then visualized with protein A-gold solution. So far several exocrine pancreatic enzymes as well as polypeptide hormones from the endocrine pancreas have been revealed by this technique.

Amylases↗
PubMed1979

Immunohistochemical localization of exocrine enzymes in normal rat pancreas.

On the basis of morphological and biochemical differences, the exocrine pancreatic tissue has been divided in peri- and teleinsular regions. In the present study the enzymatic profile of these regions has been investigated by the immunofluorescent technique using antibodies against nine pancreatic enzymes (alpha-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidase A and B, DNase and RNase A). These antibodies were specific to their antigens without cross reaction. By immunofluorescence, most acinar cells of the normal rat pancreas were positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than acinar cells in the teleinsular tissue. These data add a new parameter to describe the inhomogeneity of the exocrine pancreas.

Animals↗
PubMed1978

Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

Animals↗
PubMed1978

[Metabolism of capillary endothelium tissue and its abnormalities in diabetes mellitus (author's transl)].

Glucose is the major energetic substrate of blood capillary endothelium; aerobic glycolysis is quantitatively the most important pathway. Insulin has no direct effect on glucose utilization. The glucokinase activity of the capillary endothelium is undetectable and the plasma cell membrane is freely permeable to glucose. All pathways of glucose utilization are stimulated by a high ambient glucose concentration: intracellular sorbitol accumulation and glucose-carbon incorporation into basement membrane collagen are most sensitive to the glucose effect. During chronic hyperglycemia, the amino acid composition of the basement membrane is modified and its capacity to incorporate glucose is enhanced. The glucose induced alterations of the blood capillary metabolism may contribute to the development of the diabetic microangiopathy.

Amino Acids↗
PubMed1977

Diffusion permeability of an isolated rete mirabile.

The rete mirabile of the swimbladder of the eeel is a countercurrent exchange organ composed of alternately disposed and venous capillaries. A countercurrent perfusion of these was set up by isolating arterial input and outflow, and venous input and outflow, and perfusing each set of capillaries with oxygenated albumin-containing Krebs-Ringer bicarbonate buffer. Constant arterial infusions containing tracer albumin, inulin, 3-O-methylglucose, urea, and water were begun and tracer concentrations at the outlets of arterial and venous beds were determined. Capillary permeabilities were determined from the resulting steady state values. The method of calculation of permeability values from the data acquired in the rete involves no approximations and appears to yield unequivocal values. For the solutes, the values appear compatible with transcapillary passage by free diffusion. No evidence of increasing restriction (in comparison to the diffuse coefficients) was found over the range of molecular sizes explored. The permeability for labeled water, in relation to the solutes, was too high, and this augmentation was attributed to the larger surface available for its exchange. Despite this larger value, labeled water did not undergo flow-limited exchange. A significant barrier to its distribution is present in this capillary system.

Air Sacs↗
PubMed1975

Studies of the capillary basal lamina. I. Ultrastructure of the red body of the eel swimbladder.

The rete mirabile of the eel swimbladder is a vascular organ in which arterial andvenous capillaries alternate in regular fashion. The endothelium of the arterial capillaries is high, whereas that of the venous capillaries is thin and fenestrated. Endothelial cells and pericytes are the only cells found in the rete. A tubular systemin the arterial endothelial cells is connected with the organelles. The basal laminaeof both capillaries are similiar to those of mammalian capillaries of the same type. They present three layers of different electron density and have a high fibrillar pattern.A fine fibrillar material and dense fibrils appear in the cisternae of the rough endoplasmic reticulum and in large vacuoles of endothelial cells and pericytes: these cells areapparently involved in the synthesis of their lamina. Since the rete mirable ofthe eel swimbladder is a pure vascular preparation, it is most useful for the study of themorphology, the permeability, and the metabolism of the endothelial cells and for the analysis of the chemical structure of their basil laminae.

Air Sacs↗
PubMed1975

Studies of the capillary basal lamina. II. Preparation, chemical composition, andmetobolic properties.

Large amounts of basal laminae material can be extracted by sonic disruption from the capillary tissue of the rete mirabile of the eel swimbladder. The extracted basil laminae material has the same fibrillar pattern as that found in the intact tissue. Thechemical composition is similiar to that of other basil laminae isolated from tissues inmammals, with high content of glycine, proline, hydroxyproline, lysine, and hydroxylysine. The mono- and disaccharide units linked to the hydroxylysine are also present. Of all the pathways of glucose utilization, the 'C-glucose incorporation into the basallamina glycoproteins is the most sensitive to the medium glucose concentration. The glucosyltransferase activity seems stimulated to a plateau value of saturation when the medium glucose concentration is raised from 5 to 20 mm. The 'C-labeled amino acid incorporation into the basal laminae glycoproteins is not affected bythe medium glucose concentration. The results obtained in this study allow for a hypothesis which links the biochemical, morphologic, and functional properties of various typesof basal laminae in normal and hyperglycemic conditions.

Air Sacs↗
PubMedDate not supplied

A review of the study of protein secretion applying the protein A-gold immunocytochemical approach.

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A-gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph-node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A-gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome-like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans-side and the trans-most rigid one. Quantitative evaluations of the intensities of labeling have allowed for 1) demonstration of the high specificity of the different labelings; 2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and 3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double-labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A-gold complexes formed by gold particles of different sizes. Protein A-gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high-resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A-gold technique, which in combination with other procedures constitutes a powerful method in cell biology.

Ameloblasts↗
PubMedDate not supplied

Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity.

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.

Animals↗
PubMedDate not supplied

Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach.

Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.

Animals↗
PubMedDate not supplied

Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules.

The expression and distribution of ADP-ribosylation factor (ARF) small GTP-binding proteins in kidney tissue was examined. Various anti-ARF antibodies were raised against purified rec-ARF 1 and rec-ARF 6 and their specificity was determined. Using indirect immunofluorescence analysis of intact kidney, ARF proteins were found to be predominantly expressed in kidney tubules as compared to glomeruli. This result was further supported by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of purified human kidney glomeruli and proximal tubules. Both ARF 1 and ARF 6 were detected in purified human glomeruli and proximal tubules; however, ARF 1 was more abundant than ARF 6 in these kidney structures. Brush-border membrane vesicles (BBMV) and early endosomes (EE) derived from the receptor-mediated endocytosis pathway were isolated from purified proximal tubules of rat, dog and human kidney using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. We demonstrated that ARF 6 is associated with BBMV and with EE derived from receptor-mediated endocytosis pathway of human kidney proximal tubules. Using a combination of SDS-PAGE and quantitative enhanced chemiluminescence Western blot analysis, the quantification of the ARF 6 distribution in membrane and cytoplasmic fractions of proximal tubules was made and its predominance in membrane fractions was demonstrated. By analogy with the functional role of ARF 1 in Golgi protein transport, we suggest that ARF 6 may play an important role in the regulation of receptor-mediated endocytosis and protein reabsorption by kidney proximal tubules.

ADP-Ribosylation Factor 6↗